Partial characterization of the N－linked oligosaccharide structures on Pselectin glycoprotein ligand－1（PSGL－1）

PSGL-1, a specific ligand for P-, E- and L-selectin, was isolated from in vivo [3H]-glucosamine labeled HL-60 cells by a combination of wheat germ agglutinin-agarose and P- or E-selectin-agarose chromatography. N-linked oligosaccharides were released from the purified, denatured ligand molecule by peptide: N-glycosidase F treatment and, following separation by Sephacryl S-200 chromatography, partially characterized using lectin, ion-exchange and size-exclusion chromatography in combination with glycosidase digestions. The data obtained suggest that the N-glycans on PSGL-1 are predominantly core-fucosylated, multiantennary complex type structures with extended, poly-N-acetyllactosamine containing outer chains. A portion of the outer chains appears to be substituted with fucose indicating that the N-glycans, in addition to the O-glycans on PSGL-1, may be involved in selectin binding.     

Partial characterization of the N-linked oligosaccharide structures on P-selectin glycoprotein ligand-1 (PSGL-1)

INTroDUCTIONP-, E- and L-selectin are C-type lectins in-volved in the binding of circulating leukocytes tovarious target cellsll, 2]. The interaction(s) be-tween the selectins and the target cells is mediatedby oligosaccharide structures conjugated to specificligand molecules expressed on the taxget cell sur-faces. These ligand molecules may be recognized byone, two or all three of the selectins[1-31. AlthoughaVailable data suggest that the interaction(s) be-tween the selectins and their…     

Differences in the glycosylation of rat submandibular kallikreins

The glycosylations of five different rat submandibular kallikreins, rK1, rK2, rK7, rK9 and rK10, vacuum-blotted onto nitrocellulose membranes, have been studied by means of labelled lectins using enhanced chemiluminescence detection. The results demonstrated that individual submandibular kallikreins are not heavily glycosylated in rats, but consistently show different patterns of glycosylation. Following digestion of slot-blotted enzymes with peptide-N-glycosidase F (PNGase): binding by lectin fromLens culinaris (Man-directed) was abolished, whilst that of lectin fromMaclura pomifera (Gal1,3GalNAc-directed) persisted (but could be abolished by periodate oxidation and endo--N-acetylgalactosaminidase digestion), revealing that there are O- as well as N-linked sugar chains on the kallikreins; a novel observation for this family of enzymes. The presence of GalNAc in addition to GlcNAc, Fuc, Gal and Man, in sugar chains of rK1 was confirmed by high pH anion exchange chromatography following acid hydrolysis. Different intensities of binding by lectin fromLimax flavus (NeuNAc-directed) suggest that sialylation of individual kallikreins differs, whilst sialidase and PNGase digestions suggest that sialic acid is the terminal residue of some N-linked but not O-linked structures.     

Lectin histochemical localization of N- and O-linked oligosaccharides during the spermiogenesis of the urodele amphibian Pleurodeles waltl

The aim of this work is the characterization of the glycoconjugates of the spermatids during the spermiogenesis of the testis of an urodele amphibian, Pleurodeles waltl, by means of lectins in combination with several chemical and enzymatic procedures, in order to establish the distribution of N- and O-linked oligosaccharides in these cells. The acrosome was the most relevant lectin-labeled structure. The O-linked oligosaccharides contained DBA- and SBA-positive GalNAc, AAA-positive Fuc and PNA-positive Gal1,3GalNAc. Sialic acid was scarcely observed, the Neu5Ac2,-3Gal1,4GlcNAc sequence was found in N-linked oligosaccharides. Additionally, N-linked oligosaccharides containing HPA-positive GalNAc and AAA-positive Fuc were found. Moreover, with some lectins the acrosome showed a variable composition of the oligosaccharides in the different steps of the sperm maturation. Some residues were found only in the early steps in maturating acrosome, while others were in the later steps, showing that acrosomal glycoconjugates are modified during acrosome development in spermiogenesis. The changes observed during acrosome maturation suggest the existence of a predetermined pattern of storage of the acrosome components and a progressive compression of them.     

Reassessment of the acceptor specificity and general properties of the Lewis blood-group gene associated α-3/4-fucosyltransferase purified from human milk

The acceptor specificity and general properties of a Lewis blood-group gene associated -3/4-L-fucosyltransferase isolated from human milk have been examined at the penultimate purification stage involving affinity chromatography on GDP-hexanolamine Sepharose, and after a subsequent gel filtration step on Sephacryl S-200. Both preparations transferred fucose to theO-4 position ofN-acetylglucosamine in Type 1 (Gal1-3GlcNAc-R) acceptors and theO-3 position of glucose in lactose-based (Gal1-4Glc) oligosaccharides, and both used Type 1 sialylated compounds when the terminalN-acetylneuraminic acid was present in -2,3 linkage. The striking difference between the two preparations was in their reactivity with Type 2 (Gal1-4GlcNAc-R) chains; after Sephacryl S-200 chromatography the apparentK _M values for the -3/4- preparation with unsubstituted low-molecular-weight Type 2 oligosaccharides were considerably increased. Substitution of the terminal galactose with sialic acid in -2,3 linkage decreased theK _M values for low-molecular-weight oligosaccharides but no detectable incorporation of fucose was observed intoN-acetyllactosamine end-groups of glycoproteins withN-linked oligosaccharide chains, irrespective of the presence of sialic acid in the terminal sequences.Deceased 25 June 1991.     

O-glycosylation of the coronavirus M protein. Differential localization of sialyltransferases in N- and O-linked glycosylation.

It has previously been shown that the M (E1) glycoprotein of mouse hepatitis virus strain A59 (MHV-A59) contains only O-linked oligosaccharides and localizes to the Golgi region when expressed independently. A detailed pulse-chase analysis was made of the addition of O-linked sugars to the M protein; upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, three different electrophoretic forms could be distinguished that corresponded to the sequential acquisition of N-acetylgalactosamine (GalNAc), galactose (Gal), and sialic acid (SA). A fourth and fifth form could also be detected which we were unable to identify. Following Brefeldin A treatment, the M protein still acquired GalNAc, Gal, and SA, but the fourth and fifth forms were absent, suggesting that these modifications occur in the trans-Golgi network (TGN). In contrast, in the presence of BFA, the G protein of vesicular stomatitis virus (VSV), which contains N-linked oligosaccharides, acquired Gal and fucose but not SA. These results are consistent with earlier published data showing that Golgi compartments proximal to the TGN, but not the TGN itself, relocate to the endoplasmatic reticulum/intermediate compartment. More importantly, our data argue that, whereas addition of SA to N-linked sugars occurs in the TGN the acquisition of both SA on O-linked sugars and the addition of fucose to N-linked oligosaccharides must occur in Golgi compartments proximal to the TGN. The glycosylation of the M protein moreover indicates that it is transported to trans-Golgi and TGN. This was confirmed by electron microscopy immunocytochemistry, showing that the protein is targeted to cisternae on the trans side of the Golgi and co-localizes, at least in part, with TGN 38, a marker of the TGN, as well as with a lectin specific for sialic acid.     

Glycoconjugate histochemistry of the digestive tract of <Emphasis Type="Italic">Triturus carnifex</Emphasis> (Amphibia,Caudata)

Summary In this study, the variety of sugar residues in the gut glycoconjugates of Triturus carnifex (Amphibia, Caudata) are investigated by carbohydrate conventional histochemistry and lectin histochemistry. The oesophageal surface mucous cells contained acidic glycoconjugates, with residues of GalNAc, Gal β1,3 GalNAc and (GlcNAc β1,4) _n oligomers. The gastric surface cells mainly produced neutral glycoproteins with residues of fucose, Gal β1-3 GalNAc, Gal-αGal, and (GlcNAc β1,4) _n oligomers in N- and O-linked glycans, as the glandular mucous neck cells, with residues of mannose/glucose, GalNAc, Gal β1,3 GalNAc, (GlcNAc β1,4) _n oligomers and fucose linked α1,6 or terminal α1,3 or α1,4 in O-linked glycans. The oxynticopeptic tubulo-vesicular system contained neutral glycoproteins with N- and O-linked glycans with residues of Gal-αGal, Gal β1-3 GalNAc and (GlcNAc β1,4) _n oligomers; Fuc linked α1,2 to Gal, α1,3 to GlcNAc in (poly)lactosamine chains and α1,6 to GlcNAc in N-linked glycans. Most of these glycoproteins probably corresponds to the H⁺K⁺-ATPase β-subunit. The intestinal goblet cells contained acidic glycoconjugates, with residues of GalNAc, mannose/ glucose, (GlcNAc β1,4) _n oligomers and fucose linked α1,2 to Gal in O-linked oligosaccharides. The different composition of the mucus in the digestive tracts may be correlated with its different functions. In fact the presence of abundant sulphation of glycoconjugates, mainly in the oesophagus and intestine, probably confers resistance to bacterial enzymatic degradation of the mucus barrier.     

O-Linked glycome and proteome of high-molecular-mass proteins in human ovarian cancer ascites: Identification of sulfation, disialic acid and O-linked fucose

The O-linked glycosylation of the main acidic high-molecular-weight glycoprotein from ascites fluid from patients with ovarian cancer were analyzed. The O-linked oligosaccharides were shown to consist of mainly highly sialylated core 1 and 2 structures with a smaller amount of sulfated core 2 structures. These structures were shown to be able to be further extended into small keratan sulfate (KS)-type oligosaccharides with up to four N-acetyllactosamine units. Proteomic studies of the acidic fraction of ascites fluid from patients with ovarian cancer showed that this fraction was enriched in proteoglycans. Among them, lumican, agrin, versican and dystroglycans were potential candidates, with threonine- and serine-rich domains that could carry a significant amount of O-linked glycosylation, including also the O-linked KS. Glycomic analysis using liquid chromatography (LC)-tandem mass spectrometry (MS/MS) also showed that the disialic acid NeuAc-NeuAc- was frequently found as the terminating structure on the O-linked core 1 and 2 oligosaccharides from one ascites sample. Also, a small amount of the epidermal growth factor (EGF)-associated O-linked fucose structure Gal-GlcNAc-Fucitol was detected with and without sialic acid in the LC-MS/MS analysis. Candidate proteins containing O-linked fucose were suggested to be proteoglycan-type molecules containing the O-linked fucose EGF consensus domain.     

Evidence of regional differences in the lectin histochemistry along the ductus epididymis of the lizard,Podarcis sicula Raf

The regional difference in the carbohydrate components of the ductus epididymis epithelium of a lizard was delineated by means of 13 lectins. Basal cells expressed only N-acetylglucosamine (GlcNAc). Throughout the ductus, the secretory cells showed oligosaccharides with terminal N-acetylneuraminic acid (Neu5Ac)(2,6)galactose (Gal)/N-acetylgalactosamine (GalNAc) and internal mannose (Man) and/or glucose (Glc) in the whole cytoplasm, oligosaccharides terminating in Neu5Ac(2,6)Gal(1,3)GalNAc, Neu5Ac(2,6)Gal (1,4)GlcNAc, GalNAc, GlcNAc, and fucose (Fuc) in the supra-nuclear zone, and also glycans terminating in Neu5Ac(2,3)Gal (1,4)GlcNAc, Neu5Ac(2,6)Gal(1,3)GalNAc, Gal (1,4)GlcNAc on the luminal surface. In the caput and corpus regions, the supra-nuclear cytoplasm was characterized by terminal Gal(1,4)GlcNAc and GalNAc, the luminal surface by GalNAc and Gal. The Golgi zone, showing oligosaccharides with terminal Neu5Ac(2,3)Gal (1,4)GlcNAc, Neu5Ac(2,6)Gal (1,3)GalNAc, Neu5Ac(2,6)Gal (1,4)GlcNAc, and internal GlcNAc, expressed terminal Gal (1,4)GlcNAc and GalNAc in the caput, and terminal GalNAc in the corpus. The granules showed all the investigated carbohydrates in their peripheral zone except terminal GalNAc and Fuc, whereas internal Man/Glc and terminal Gal were expressed in the central core, and Fuc throughout the ductus, terminal GlcNAc in the caput and corpus, and terminal GalNAc only in the corpus.     

GalNAc moieties in O-linked oligosaccharides of the primordial germ cells of Xenopus embryos

Glycoconjugates could play a role in cell adhesion and migration mechanisms, including the locomotive movements of the primordial germ cells (PGCs) during the development of the embryo. In the present work, we have studied by lectin histochemistry the presence of N-acetylgalactosamine (GalNAc) in the glycans of the Xenopus PGCs, as a first approach to identifying their glycoconjugates which could be involved in the migration mechanism. The PGCs were negative for three of the GalNAc-binding lectins employed (from soybean, SBA; from lima bean, LBA; and from snail, HPA). However, when sialic acid (NeuAc) was previously removed by acid hydrolysis, SBA and HPA, but not LBA, labeled the PGCs, except if the staining was combined with the beta-elimination procedure. This suggests the presence of GalNAc alpha(1,3)-linked to galactose (Gal) in O-linked oligosaccharides, in a subterminal position to NeuAc. As the PGCs were always negative for LBA, the absence of fucose alpha(1,2)-linked to subterminal Gal is suggested. With the lectin from horse gram (DBA), the PGCs were stained, although beta-elimination turned the cells negative and acid hydrolysis increased the labeling, suggesting that GalNAc(alpha)(1,3)GalNAc was in O-linked glycans in terminal and subterminal to NeuAc position.     

Isolation and characterization of a Forssman antigen-binding lectin from velvet bean (Mucuna derringiana) seeds

A Forssman antigen (GalNAc1-3GalNAc1-3Gal1-4Gal1-4Glc1-1Cer)-binding lectin has been purified from velvet bean (Mucuna derringiana) seeds by a combination of affinity chromatography and reversed phase HPLC. This lectin agglutinates both native and trypsin-treated sheep erythrocytes as well as trypsinized rabbit erythrocytes, but neither native rabbit nor human erythrocytes, irrespective of blood group type. SDS-PAGE and gel filtration chromatography reveal the lectin to be a homodimer consisting of two 54 kDa subunits linked by non-covalent bonds. The results obtained by quantitative precipitation, haemagglutination inhibition and TLC overlay assays indicate that theMucuna lectin specifically recognizes Forssman antigen and Forssman disaccharide (GalNAc1-3GalNAc)-related structures. Abbreviations: The abbreviations and the trivial names used are: AH, 6-aminohexyl; BSA, bovine serum albumin; Cer, ceramide; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis; PBS, 10mm phosphate-buffered saline, pH 7,2, containing 0.15m NaCl; PMSF, phenyl methyl sulfonyl fluoride; SDS, sodium dodecyl sulphate; TFA, trifluoroacetic acid; TBS, 20mm tris-buffered saline, pH 7.2; TLC, thin-layer chromatography; A disaccharide, GalNAc1-3Gal; A trisaccharide, GalNAc1-3[Fuc1-2]Gal; Forssman disaccharide, GalNAc1-3GalNAc; CDH (ceramide dihexoside or lactosyl ceramide) Gal1-4Glc1-1Cer (LacCer); CTH (ceramide trihexoside or globotriosyl ceramide), Gal1-4Gal1-4Glc1-1Cer (GbOse₃Cer or Gb₃); globoside (globotetraosyl ceramide), GalNAc1-3Gal1-4Gal1-4Glc1-1Cer (GbOse₄Cer or Gb₄); Forssman antigen (globopentaosyl ceramide), GalNAc1-3GalNAc1-3Gal1-4Gal1-4Glc1-1Cer (GbOse₅Cer).     

Structural requirements for the binding of oligosaccharides to immobilized lectin ofErythrina variegata (Linn) var.Orientalis

The structural requirements for the interaction of asparagine-linked oligosaccharide moieties of glycoproteins withErythrina variegata agglutinin (EVA) were investigated by means of affinity chromatography on an EVA-Sepharose column. Some of the branched poly-N-acetyllactosamine-type oligosaccharides obtained from human erythrocyte band 3 glycoprotein were found to show high affinity to EVA-Sepharose, whereas complex-type oligosaccharides were shown to have low affinity. Hybrid type, oligomannose-type and unbranched poly-N-acetyllactosamine-type oligosaccharides bound very little or not at all to EVA-Sepharose. To further study the carbohydrate-binding specificity of this lectin, we investigated the interaction of immobilized EVA and oligosaccharide fragments obtained through partial hydrolysis from branched poly-N-acetyllactosamine-type oligosaccharides. Branched poly-N-acetyllactosamine-type oligosaccharides were subjected to limited hydrolysis with 0.1% trifluoroacetic acid at 100°C for 40 min and then separated on an amino-bonded silica column. One of pentasaccharides thus prepared strongly bound to the EVA-Sepharose column. Structural analysis of this pentasaccharide showed that the Gal1-4GlcNAc1-3(Gal1-4GlcNAc1-6)Gal sugar sequence, which is an l-antigen determinant, was essential for the high affinity binding of the oligosaccharides to the EVA-Sepharose column.Abbreviations EVA Erythrina variegata agglutinin - WGA wheat germ agglutinin - STA potato lectin - LEA tomato lectin - DSA Datura stramonium agglutinin - PBS 0.01 M sodium phosphate buffer, pH 7.3, containing 0.15 M NaCl - Galol galactitol     

The substrate specificity of the enzyme Endo-α-N-acetyl-D-galactosaminidase from Diplococcus pneumonia

The substrate specificity of the enzyme endo-α-N-acetyl-D-galactosaminidase from Diplococcus pneumonia was re-examined using bovine submaxillary mucin and remodelled antifreeze glycoprotein as substrates. Incubation with desialylated bovine submaxillary mucin, which contains six O-linked core types, indicated that the disaccharide Galβ1-3GalNAc, which is present in very small amount, was the only glycan released, while the disaccharide GlcNAcβ1-3GalNAc, which is the major structure present, and other disaccharides, were not released. To test whether the core disaccharide Galβ1-3GalNAc with sialic acid linked α2-3 to the Gal or linked α2-6 to the GalNAc was released, the enzyme was incubated with remodelled antifreeze glycoprotein containing (1) [3H]NeuAcα2-3Galβ1-3GalNAc and (2) Galβ1-3[[14C]NeuAcα2-6]GalNAc as substrates. No NeuAc-containing trisaccharide was released. These results serve to clarify the doubts of many researchers regarding the activity of this enzyme on some newly-described core types and on sialylated substrates. This revised version was published online in November 2006 with corrections to the Cover Date.     

Lectin affinity chromatography of proteins bearing O-linked oligosaccharides: application of jacalin-agarose.

The lectin jacalin immobilized on agarose was found to bind a variety of glycoproteins known to contain typical O-linked oligosaccharides, including human IgA, C1 inhibitor, chorionic gonadotropin, plasminogen, bovine protein Z, bovine coagulation factor X, and fetuin. These proteins were eluted from columns of jacalin-agarose specifically by alpha-galactopyranosides such as melibiose and alpha-methylgalactopyranoside but not by lactose or other sugars. Treatment of asialofetuin with endo--alpha--N--acetylgalactosaminidase eliminated its affinity for the lectin column, and other proteins known to contain only N-linked oligosaccharides such as ovalbumin, transferrin, and alpha 1-acid glycoprotein were not retained by the lectin. Binding of proteins with O-linked oligosaccharides to the lectin column did not require divalent cations and was affected little by changes in pH and ionic strength over a wide range. Virtually all of the glycosidically linked oligosaccharides of fetuin, chorionic gonadotropin, and plasminogen are known to be sialated. Thus, binding of these glycoproteins to jacalin, which is known to have affinity for the core disaccharide, 1-beta-galactopyranosyl-3-(alpha-2-acetamido-2-deoxygalactopyranoside ), in O-linked oligosaccharides of these proteins, was not prevented by the presence of sialic acids. Affinity of oligosaccharides for jacalin did appear to be reduced by occurrence of sialic acids as it was found that higher concentrations of melibiose were required to elute asialofetuin than fetuin from jacalin-agarose. Results of the present study indicate that affinity chromatography using this lectin is a widely applicable technique for identifying and purifying proteins bearing O-linked oligosaccharides.     

Lectin cytochemical evaluation of somatosensory neurons and their peripheral and central processes in rat and man

Summary Paraffin sections of the trigeminal nerve root of the rat, and human spinal nerve root and trigeminal ganglion were stained with a battery of lectin-horseradish peroxidase conjugates to localize and characterize glycoconjugate (GC) in situ. In the rat the myelin sheath of the peripheral segment contained GC with sialic acid most probably linked to the penultinate disaccharide galactose(1 4)-N-acetylglucosamine (Gal(1 )-GlcNAc), and complex type N-glycosidic side chains. The myelin sheath in the central segment differed in containing little if any of the GC named above and in containing GC with terminal -Gal linked to N-acetylgalactosamine (GalNAc), terminal GalNAc and fucose. Schwann cells stained for GC with GlcNAc or mannose whereas oligodendroglia stained for GC with the terminal disaccharide Gal-(1 3)-GalNAc and N-glycosidic side chains, especially in presumed Golgi zones, but also in processes continued as the outer myelin sheath. The human myelin sheath in the central segment differed from that of the rat in not staining with lectins specific for fucose and terminal GalNAc. Sialic acid and terminal -Gal were seen in the human central segment but these sugars appeared to bind to astroglial structures rather than to the myelin sheath as in the rat. Astrocytes in both rat and man were stained by two fucose-binding lectins. Several lectins revealed affinity for GC in the neurilemmal sheath, and staining of this structure was stronger in the human specimens. Neurons in the human trigeminal ganglion ranged from unstained to strongly positive for fucoconjugate in cytoplasmic bodies and plasmalemma. Positive ganglion cells gave rise to unmyelinated fibers which also stained for fucoconjugate. Remak fibers and their extensions into the substantia gelatinosa of the human spinal cord stained strongly for content of fucose.The stronger lectin affinity for N-glycosidic core sugars in the peripheral as compared with the central segment suggests that lectins localize P_o protein in peripheral myelin. The reactivity for several sugars in the central segment can possibly be attributed to myelin-associated glycoprotein (MAG) of central myelin, but lectin staining for GalNAc shows in addition a biochemically unrecognized GC with O-glycosidic linked oligosaccharides in myelin. The lectin cytochemistry indicates that the 170 K Dalton glycoprotein with PNA affinity obtained from rat sciatic nerves occurs in nodes of Ranvier.This research was supported by NIH Grants AM-10956, HL-29775 and United Health and Medical Research Foundation of South Carolina, Inc. Grant No. 79     

Lectin histochemical identification of the carbohydrate moieties on N- and O-linked oligosaccharides in the duct cells of the testis of an amphibian urodele, the spanish newt (Pleurodeles waltl)

The aim of the present work was to study the carbohydrate moieties present on N- and O-linked oligosaccharides of duct cells of a urodele amphibian testis, by means of lectin histochemistry. It was found that duct cells have a carbohydrate composition that includes ensuremath (1,3)-, (1,4)- or (1,6)-linked Fuc and Man on N-linked oligosaccharides, Gal and GlcNAc on O-linked oligosaccharides, and DBA-positive GalNAc, (1,2)-linked Fuc and Neu5Ac(2,3)Gal (1,4)GlcNAc on both N- and O-linked oligosaccharides. All the duct cells showed the same lectin labelling pattern, the only exception being some sparse duct cells that showed the sequence Neu5Ac(2,6)Gal/GalNAc. The possible roles of duct cells in sperm maturation and the hypothesis for a common origin of duct and follicle (Sertoli) cells in the urodele testis are discussed.     

Differential lectin binding patterns in the oviductal ampulla of the horse during oestrus

We investigated the oligosaccharide sequence of glycoconjugates, mainly sialoglycoconjugates, in the horse oviductal ampulla during oestrus by means of lectin and pre-lectin methods such as the KOH-neuraminidase procedure to remove sialic acid residues and incubation with N-glycosidase F to cleave N-linked glycans. Ciliated cells displayed N-linked oligosaccharides throughout the cytoplasm. The cilia glycocalyx expressed both N- and O-linked (mucin-type) oligosaccharides, both showing a high variety of terminal sequences. In the most non-ciliated cells, the whole cytoplasm contained N-linked oligosaccharides with terminal alphaGal as well as mucin-type glycans with terminal Forssman pentasaccharides. In a few scattered non-ciliated cells, the whole cytoplasm displayed sialylated N-linked oligosaccharides with terminal Neu5Ac-GalNAc and O-linked glycans terminating with neutral and/or alphaGalNAc, Neu5Ac alpha2,6Gal/GalNAc, Neu5AcGal beta1,3GalNAc. Supra-nuclear granules, probably Golgi zones, of non-ciliated cells showed mainly O-linked glycans rich in sialic acid residues. The luminal surface of non-ciliated cells showed N-linked oligosaccharides, containing terminal/internal alphaMan/alphaGlc, betaGlcNAc and terminal alphaGal, as well as mucin-type oligosaccharides terminating with a large variety of either neutral saccharides or sialylated sequences. Apical protrusions containing O-linked oligosaccharides with terminal Forssman pentasaccharide, Neu5Ac-Gal beta1,4GlcNAc, Neu5Ac-GalNAc were seen in non-ciliated cells scattered along the epithelium. These findings show the presence of sialoglycoconjugates in the oviductal ampulla epithelium of the mare and the existence of different lectin binding profiles between ciliated and non-ciliated (secretory) cells, as well as the presence of non-ciliated cell sub-types which might determine functional differences along the ampullary epithelium of mare oviduct.     

Specificity studies of an antibody developed against a mucin-type glycoprotein

The specificity of a new anti-epiglycanin antibody (AE-3) which recognizes a mucin-type glycoprotein, the Human Carcinoma Antigen, found in the blood of patients with carcinomas, was studied. Information regarding the chemical nature of the antibody binding site was obtained by altering the structure of epiglycanin by chemical or enzymic means and testing the product in a competitive binding assay for inhibition of the binding of AE-3 to epiglycanin. The need for a high molecular weight antigen containing clustered T disaccharide, Gal1-3GalNAc, was demonstrated. The specificity was further explored by inhibition studies with glycopeptides having one to three mono- to disaccharides. The results were interpreted using computer graphics molecular modeling which predicted the specific recognition of hydroxyl groups on oligosaccharides on adjacent amino acids. Thus T antigen O-linked glycopeptide tumour markers can be designed to be distinguished by antibodies by the amount of clustering of their oligosaccharides.     

Deaminated neuraminic acid-rich glycoprotein of rainbow trout egg vitelline envelope. Occurrence of a novel alpha-2,8-linked oligo(deaminated neuraminic acid) structure in O-linked glycan chains

Deaminated neuraminic acid-rich glycoprotein (KDN-gp), first found and isolated from the vitelline envelope of rainbow trout eggs (Inoue, S., Kanamori, A., Kitajima, K., and Inoue, Y. (1988) Biochem. Biophys. Res. Commun. 153, 172-176), has been found to contain a number of O-linked glycan. Oligosaccharides were released by alkaline borohydride treatment of KDN-gp. Following fractionation by DEAE-Sephadex A-25 and thin-layer chromatography, a series of acidic oligosaccharides were obtained and analyzed for their chemical structures. The structure is based on composition analysis, methylation analysis, alkali-catalyzed "peeling" reactions, periodate oxidation, 400-MHz one- and two-dimensional 1H NMR spectroscopy, and molecular secondary ion mass spectrometry. The O-linked oligosaccharides isolated from KDN-gp have been shown to contain a common core trisaccharide Gal beta 1-3GalNAc alpha 1-3GalNAc in which the terminal Gal residue is blocked by a single residue of deaminated neuraminic acid (KDN) and the proximal GalNAc residue is substituted by alpha-2,8-linked oligo(KDN) chains. Structures of KDN-oligosaccharide chains in the glycoprotein are novel and expressed by the following general formula, where n = 0-5, for which data are available. [formula: see text]     

Identification of the oligosaccharide structures of human coagulation factor X activation peptide at each glycosylation site

Human blood coagulation factor X has two N-linked oligosaccharides at Asn³⁹ and Asn⁴⁹ residues and two O-linked oligosaccharides at Thr¹⁷ and Thr²⁹ residues in the region of the factorX activationpeptide (XAP) which is cleaved off during its activation by factor IXa. We determined the structure of oligosaccharides in the XAP region of human factor X. Four glycopeptides each containing a glycosylation site were isolated by digestion of XAP with endoproteinase Asp-N followed by reversed-phase HPLC. N-linked oligosaccharides released from the glycopeptides by glycoamidase A digestion were derivatized with 2-aminopyridine. Pyridylamino(PA)-oligosaccharides were separated by HPLC into neutral and sialyl oligosaccharides using an anion-exchange column. Structures of oligosaccharides and their contents at each glycosylation site were determined by a two-dimensional sugar mapping method. The contents of the neutral oligosaccharides at Asn³⁹ and Asn⁴⁹ residues were 32.5% and 30.0%, respectively. Six neutral and twelve monosialyl oligosaccharides isolated from both N-linked glycosylation sites showed similar elution profiles composed of bi-, tri-and tetra-antennary complex type oligosaccharides. The predominant component in neutral oligosaccharides was biantennary without a fucose residue. Two major monosialyl oligosaccharides were also biantennary without fucose and with a Neu5Ac-26 residue. In addition, the structures of O-linked oligosaccharides at Thr¹⁷ and Thr²⁹ residues were suggested to be disialylated Gal/3GalNAc sequences by their component analyses.Abbreviations Gal d-galactose - GlcNAc N-acetyl-d-glucosamine - Man d-mannose - HPLC high-performance liquid chromatography - NDV Newcastle disease virus - Neu5Ac 5-N-acetylneuraminic acid - ODS octadecylsilyl - PA pyridylamino - RVV-X Russell's viper venom factor X activator - TBS Tris-buffered saline - XAP factor X activation peptide.